Purification and enzymic properties of the fructosyltransferase of Streptococcus salivarius ATCC 25975

Abstract

The recombinant fructosyltransferase (Ftf) of Streptococcus salivarius was expressed in Escherichia coli and purified to electrophoretic homogeneity after a combination of adsorption, ion-exchange and gel-filtration chromatography. The N-terminal signal sequence of the Ftf was removed by E. coli at the same site as in its natural host. The purified Ftf exhibited maximum activity at pH 6.0 and 37 degrees C, was activated by Ca2+, but inhibited by the metal ions Cu2+, Zn2+, Hg2+ and Fe3+. The enzyme catalysed the transfer of the fructosyl moiety of sucrose to a number of acceptors, including water, glucose and sucrose via a Ping Pong mechanism involving a fructosyl-enzyme intermediate. While this mechanism of catalysis is utilized by the levansucrases of Bacillus subtilis and Acetobacter diazotrophicus and the values of the kinetic constants for the three enzymes are similar, sucrose was a far more efficient fructosyl-acceptor for the Ftf of S. salivarius than for the two other enzymes.

Identification and functional characterization of levS, a gene encoding for a levansucrase from Leuconostoc mesenteroides NRRL B-512 F

“…A low residual activity was obtained in solution containing up to 80 μM of EDTA, but it is completely lost at 1 mM EDTA. Similar results have been reported for LS from S. salivarius (Song and Jacques, 1999), where 1 mM EDTA reduced the enzyme activity by 83%. However, levansucrases from L. reuteri (Ozimek et al, 2005) and L. mesenteroides (MIFT) (Kang et al, 2005) are not strongly influenced losing only 10-20% of the initial activity in the presence of 1 mM EDTA.…”

Kinetic properties of an inulosucrase from Lactobacillus reuteri 121

“…Inu displays a temperature optimum at 50°C and non‐Michaelis–Menten type of kinetics for the transferase reaction. For levansucrases [6–8] and the S. mutans inulosucrase [3], temperature optima of 37°C have been reported. The Inu pH optimum of 5.0–5.5 is comparable to the values reported for other FTFs.…”

Microbial fructosyltransferases and the role of fructans

“…1974), Gluconacetobacter diazotrophicus (Hernández et al. 1995) and Streptococcus salivarius (Song and Jacques 1999).…”